Biofermentation is an important technology for the biocatalytic conversion of renewable resources. Microbial products produced by means of biofermentation include amino acids, ethanol, and antibiotics. The biofermentative production and commercialization of a few chemicals has been reported (W. Crueger and A. Crueger, Biotechnology: A Textbook of Industrial Microbiology, Sinauer Associates: Sunderland, Mass., pp 124–174 (1990); B. Atkinson and F. Mavituna, Biochemical Engineering and Biotechnology Handbook, 2nd ed.; Stockton Press: New York, pp 243–364 (1991)). Biocatalytic processes, however, frequently suffer from several well-known limitations compared to synthetic processes. These limitations include 1) a relatively small range of products; 2) low yields, titers, and productivities; and 3) difficulty recovering and purifying products from aqueous solutions.
The productivity of a biocatalytic process can be interfered with by accumulating product in several ways. At the biochemical level, feedback inhibition from product accumulation can limit productivity either because of inhibitory effects (which may be reversible) or toxicity effects (which can ultimately kill the microorganism or irreversibly inactivate its biocatalytic components). With regard to cell physiology, accumulating product can deleteriously affect growth rate. Chemical and physical effects (accumulating by-products; pH changes) can also interfere with the productivity of the biocatalyst.
Products of biocatalytic processes may also be lost from the system by 1) degrading from further interaction with the biocatalyst, 2) from environmental conditions, or 3) from uncontrolled removal from the system (i.e., from evaporation).
Although metabolic engineering alone can address some of these limitations, integrating upstream metabolic engineering (i.e., product synthesis) and downstream bioprocess engineering (i.e., product separation and process design) is critical to realize significant value from industrial biofermentations.
In situ product removal (ISPR) methodologies are a family of techniques in which a target molecule in the biofermentation (either a biofermentation product or other specific byproducts) is removed as it is synthesized during at least a portion of the biofermentation process (reviewed in Chauhan et al., ChemTech 27: 26–30 (1997); and Freeman et al., Biotechnology 11: 1007–1012 (1993)). Since a variety of separation principles can be used for ISPR, including those based on different volatility, solubility, size, density, charge, or specific elements (or combinations of these methods), ISPR techniques have wide applicability. A number of ISPR techniques have been integrated into biocatalytic processes based upon Amberlite XAD resins, continuous precipitation, reactive solvent extraction followed by simultaneous extraction and back extraction, and an extractive hollow-fiber membrane reactor (Lye et al., Trends in Biotechnology 17:395–402 (1999)).
A key challenge to successful use of ISPR in biofermentations is how to apply separation technology to large-scale industrial processes in a cost- and time-effective manner that increases productivity. One bioengineering factor that significantly affects productivity of a biofermentation recovery and purification system is the mode of process operation. Those skilled in the art know that it is generally more cost- and time-effective to rely on a continuous separation method versus a purely batch process.
U.S. Pat. No. 6,114,157 illustrates the challenges of using ISPR techniques cost- and time-effectively. The patent describes an ISPR method for increasing total production of 4-hydroxybenzoic acid (PHB) by biofermentation. Genetically engineered E. coli cells produce PHB during the biofermentation. For at least a portion of the biofermentation, the biofermentation broth passes through a bed of anion exchange beads in an upwards direction. The biofermentation medium depleted of PHB then returns to the biofermentor. This process cycled the entire culture volume through the beads every ten minutes, with no need for media replacement. When the anion exchange beads became saturated, the biofermentation was stopped, and PHB was extracted from the resin with acidic ethanol or sodium chloride in a water/ethanol mixture.
Although U.S. Pat. No. 6,114,157 does disclose ISPR separation of a biofermentation product, with media recycling back to the biofermentor, the effectiveness of the method is limited by its reliance on expanded bed adsorption. Expanded bed adsorption is not a continuous process, but instead requires a strategy of “load and elute” which means increased process time for isolating product with multi-step processes. The method is therefore not cost- or time-efficient for large-scale commercial applications. For example, adsorbent beads require a water rinse before elution, and may also require reconditioning with phosphate buffer before each reuse. U.S. Pat. No. 6,114,157 discloses an expanded bed adsorber that is approximately equivalent to half the size of the biofermentor. In industrial practice, this would add significantly to the commercial investment. Further, non-efficient use of resin and eluent would significantly increase the cost for large-scale commercial applications. Specifically, the volume of ethanol to elute product is large, relative to the amount of product recovered, and the ethanol may gradually evaporate during regeneration of the expanded beds. The eluent also requires one molar equivalent of trifluoroacetic acid, a costly additive. Additionally, biofermentation with microorganisms having high respiration rates could not use expanded bed adsorption for product separation, since dissolved oxygen content in the expanded bed would be low for extended periods. This would be severely detrimental to microorganism viability. In addition to limitations inherent in the use of expanded bed adsorption, U.S. Pat. No. 6,114,157 does not describe a process for separating neutrally charged products, for which ion exchange methods are not generally effective.
One ISPR separation technology that is operative and cost- and time-efficient at the process or production scale (in contrast to the analytical or preparative scale) is simulated moving bed (SMB) chromatography. This technique is a continuous chromatographic process, which relies on counter-current chromatography or simulated counter-current chromatography to achieve a separation (LeVan, M. D. In Perry's Chemical Engineers' Handbook, 7th Edition; p. 16–60; Perry, R. H., D. W. Green, and J. O. Maloney (Eds.); McGraw-Hill: New York; (1997)). This is widely recognized as a solvent-saving, efficient technology (U.S. Pat. No. 4,851,573, column 11). The operating principles for SMB chromatography are described in U.S. Pat. No. 2,985,589. Use of SMB chromatography is now an established technique for production-scale applications. Other process chromatography methods include, but are not limited to, cross-flow chromatography and radial chromatography. However, as currently practiced, process chromatography methods are unable to selectively separate biofermentation products and recycle the other media components to the biofermentor. This occurs because a portion of the eluent required to drive chromatographic separation would accumulate in the biofermentor, reducing its capacity. Although the duration of the biofermentation can be lengthened to increase productivity, continual media replacement increases costs for large-scale manufacturing. This is especially true when the biofermentation media contains costly cofactors and other required components for the biocatalysts' growth.
Thus, the problem to be solved is the lack of a bioprocess engineering method to selectively remove interfering target molecules produced during the biofermentation reaction without adding fresh media or accumulating eluent in the biofermentor. Ideally, the bioprocess engineering method would: 1) remove the target molecule causing toxicity or feedback inhibition of the bioprocess, 2) alleviate replacement of media withdrawn from the biofermentor for ISPR, and 3) prevent eluent accumulation in the biofermentor. Such a technique would greatly improve bioprocess performance, increasing the total production rate of the biocatalyst. As a result, higher capital productivity and potentially higher reaction yields would be achieved.